Sandbox713

This sandbox is in use until August 1, 2011 for UMass Chemistry 423. Others please do not edit this page. Thanks! Chem423 Team Projects: Understanding Drug Mechanisms

Group Members
Inna Brockman, Robert Nathan, Sarena Horava, Nick Cadirov

p38 kinase
P38 kinase belongs to one of the four subgroups of mitogen-activated protein (MAP) kinases. MAP kinases respond to extracellular stimuli by a signaling cascade leading to intracellular responses. Therefore, MAP kinases function to regulate fundamental cellular processes [1-5].

The p38 subgroup consists of four isoforms: p38α, p38β, p38γ, and p38δ [1-3]. Of these isoforms, p38α and p38β have the most similar amino acid sequences and both forms are expressed in most cell types. P38γ is mostly found in skeletal muscle, while p38δ is only found in the skin, small intestine, pancreas, and kidney [1, 2]. Since p38α was first discovered, most publications focus on this isoform and refer to p38α as p38 [2]. However, all four isoforms have the Thr-Glu-Tyr dual phosphorylation site in the regulatory loop. The substrate specificity of p38 is controlled Glu residue in this dual phosphorylation motif and the length of the loop [1]. This specificity is important for the signal cascade generated in response to stimuli.

Operating as a signal transduction mediator, p38 is activiated by both stress and mitogen stimuli. Environmental stress, particularly UV radiation and osmotic shock, cause an increase activity level of p38. Also, p38 is activated by pro-inflammatory cytokines, especially tumor necrosis factor (TNF) and interleukin-1 (IL1) [3]. However, activation of p38 depends on both the stimulus and the cell type [1]. Dual phosphorylation on the Thr and Tyr is necessary for p38 activation. This dual phosphorylation motif is common in all members of the MAP kinase family. Upstream kinases, which are the MAP kinase kinases (mkks), are responsible for p38 activation [1-3]. Due to selective activation, each p38 isoform requires distinct mkks. Further upstream activators of the MKK/p38 pathway are widely diversified. This cascade accounts for the various stimuli that lead to activating the p38 pathway [1]. Dephosphorylation by dual phosphatases is responsible for the major of the downregulation of p38 [1,3].

The activation of p38 pathway leads to the activation of downstream substrates, such as protein kinases and transcription factors. The p38 pathway regulates close to a hundred genes. P38 is associated with the expression of many cytokines, transcription factors, and cell surface receptors [1]. Various proteins that control transcription and translation are targeted, either directly or indirectly, by p38 kinases. Biological results of p38 activation include inflammation, apoptosis, cell cycle, and cell differentiation [1-3]. However, the role of p38 is specific to cell type [1].

The role of the p38 pathway in cellular inflammation places p38 as a key therapeutic target for inflammatory diseases, cancer, and other diseases. Therefore, p38 inhibitors are key therapeutic agents for the treatment of such diseases. Pyridinyl imidazoles, especially SB203580 (ligand shown in the Jmol diagram), inhibit the catalytic activity of p38 by binding to the ATP site [5]. The ATP binding site provides specificity necessary for highly selective pyridinyl imidazole inhibitors. The inhibitors for p38 do not bind to structurally similar MAP kinases. Other structural factors, such an unique pocket in p38 for the fluorophenyl ring of some pyridinyl imidazole inhibitors, contribute to the selectively of the inhibitor [4]. SB203580 and related inhibitors binds with about equal affinity to both the activated and inactivated forms of p38 kinase. Therefore, binding of the inhibitor can lock p38 into an inactivated conformation.

Gleevec is a brand name drug that targets p38. Gleevec, which is imatinib mesylate, is an inhibitor of p38. Imatinib mesylate, chemically designated as 4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide methanesulfonate [6], is structurally similar to SB203580, 4-[5-(4-fluoro-phenyl)-2-(4-methanesulfinyl-phenyl)-3h-imidazol-4-yl]-pyridine [7]. Gleevec is used to treat certain cancers, including chronic myeloid leukemia, gastrointestinal stromal tumors, and myelodysplastic/myeloproliferative diseases. Gleevec inhibits p38, preventing the proliferation of cancer cells [6].

Overall Structure
Kinase p38 is a single 351 amino acid polypeptide chain made up of 10 alpha helixes and 10 beta strands. Kinase p38 is composed of two domains. The first is a 135 residue N-terminal domain and the second a 225 residue C-terminal domain. The beta strands in light blue form antiparallel beta sheets and are located mainly towards the N-terminus while the alpha helixes in green are located mainly towards the C-terminus. In this rainbow representation of Kinase p38 the N and C termini are found at the top of the protein. The catalytic site where the drug binds is located between the two domains.

Drug Binding Site
The p38 kinase-SB2 complex binding chemistry is analyzed in this section. The ligand is connected to the binding site by a hydrogen bond with Met109 and the cyclopropylmethyl group binds to the phosphate-binding ribbon in a depression formed by Val30 and Val38. The complex is also stabilized by more distant bonds contributing from Lys53 and Val105. The conformation of the phosphate-binding ribbon of the p38 kinase changes significantly in order to bind with SB2.

Additional Features
Kinase p38 has a large lobe and small lobe between (also known as domains), which the inhibitors can bind. Blocking p38 kinase may be an valuable way of treating many inflammatory diseases. The pyridinylimidazole inhibitors bind in the ATP binding site, making it competitive against ATP. There has also been observed an allosteric binding site in the Asp-Phe-Gly motif in the active site on p38 kinase for a “diaryl urea class of highly potent and selective inhibitors”. These inhibitors have a new binding mechanism, which does not directly compete with ATP. BIRB 796 is an inhibitor, which has a 12,000-fold increase in binding affinity compared to the previous inhibitor. Structural changes that made such an increase in binding affinity are: methyl substituent on pyrazole ring replaced with tolyl group, chlorophenyl group replaced with naphthyl moiety, and addition of ethoxymorpholine subsituent on paththyl ring. Tolyl has hydrophobic interactions with with side chains of Glu 71 side chain. Glu 71 has one hydrogen bond with the NH on urea. This new conformation is better for supporting the hydrophobic interactions on the tolyl group with the inhibitor. Information about the structure of p38 kinase and its interaction with inhibitors is useful for research about reducing inflammation and diseases such as rheumatoid arthritis. New inhibitors are being designed an optimized to help fight inflammation and other diseases such as rheumatoid arthritis.

Credits
Introduction - Sarena Horava

Overall Structure - Robert Nathan

Drug Binding Site - Nick Cadirov

Additional Features - Inna Brockman